DNA fragments, probes and amplification primers of the 65 kD antigen of mycobacteria

Abstract

Nucleotide fragment of DNA, whose nucleotide sequence is included in the gene of the species of the genus Mycobacterium, coding for the 65-kD mycobacterial antigen, containing regions which are homologous in practically all species of the genus Mycobacterium, and at least one species-specific variable region, characterized in that said fragment is chosen from fragments whose nucleotide sequences possess at least 70% homology, and preferably at least 85% homology, with a predetermined sequence or its complementary sequence, said predetermined sequence beginning at nucleotide 438 and ending at nucleotide 751 of said gene coding for said antigen of all species of mycobacteria except for the species M. tuberculosis, M. bovis BCG, M. avium, M. paratuberculosis, M. fortuitum, M. malmoense, M. leprae, M. kansaii and M. marinurn.

We claim: 
     
       1. A nucleotide fragment consisting of nucleotides 438 to 751 of a gene coding for a 65-kD mycobacterial antigen or a fully complementary sequence. 
     
     
       2. The fragment of claim 1, wherein said fragment consists of SEQ ID NO: 23 or its fully complementary sequence. 
     
     
       3. A nucleotide fragment consisting of a nucleotide sequence that is at least 85% homologous with a predetermined sequence or its complementary sequence, said predetermined sequence consisting of nucleotides 438 to 751 of a gene coding for a 65-kD mycobacterial antigen, wherein said fragment comprises a nucleotide sequence selected from the group of sequences consisting of SEQ ID NO: 1, SEQ ID: NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; SEQ ID NO: 7; and their respective fully complementary sequences and said fragment contains at least one region which is homologous in practically all species of Mycobacterium and at least on species-specific variable region. 
     
     
       4. A specific primer for the amplification of a genomic DNA of Mycobacterium, wherein said primer is capable of specifically hybridizing to constant region of a single stranded fragment defined in claim 1 and comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 8; SEQ ID NO: 9; and their respective fully complementary sequences. 
     
     
       5. A probe specific for M. tuberculosis complex and capable of specifically hybridizing a region of a single-stranded fragment defined in claim 1 of a gene of a species of said complex that codes for a 65-kD antigen, said probe comprising a nucleotide sequence having at least 85% homology with the sequence of SEQ ID NO: 12 or a fully complementary sequence of SEQ ID NO: 12. 
     
     
       6. The probe of claim 5, said probe comprising a nucleotide sequence of SEQ ID NO: 12 or a fully complementary sequence of SEQ ID NO: 12. 
     
     
       7. A probe specific for M. avium-intracellulare complex and capable of specifically hybridizing a region of a single-stranded fragment defined in claim 1 of a gene of a species of said complex that codes for a 65-kD antigen, said probe comprising a nucleotide sequence having at least 85% homology with a sequence selected from the group of sequences consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20; SEQ ID NO: 21; and their respective fully complementary sequences. 
     
     
       8. The probe of claim 7, said probe comprising a nucleotide sequence selected from the group of sequences consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20; SEQ ID NO: 21; and their respective fully complementary sequences. 
     
     
       9. A probe consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 10; SEQ ID NO: 11; and their respective fully complementary sequences.